Imidazoline-based nitroxide radicals are often used as spin probes for medium acidity and polarity in different systems. In this work, using the density functional theory (DFT) approach, we have studied how physicochemical characteristics (geometry, atomic charges, and electron spin density distribution) of pH-sensitive spin-label 4-amino-2,2,5,5-tetramethyl-3-imidazoline-N-oxyl (ATI) depend on protonation and aqueous surroundings. Our calculations demonstrate that ATI protonation should occur at the nitrogen atom of the imidazoline ring rather than at the amino group. Protonation of ATI leads to a decrease in a spin density on the nitrogen atom of the nitroxide fragment >N-O-.. For simulation of ATI hydration effects, we have constructed a water shell around a spin-label molecule by means of gradual (step-by-step) surrounding of ATI with water molecules (n(H2O) = 2-41). Calculated spin density on the nitrogen atom of the nitroxide fragment increased with an extension of a water shell around ATI. Both protonation and hydration of ATI caused certain changes in calculated geometric parameters (bond lengths and valence angles). Investigating how structural and energy parameters of a system ATI-(H2O)(n) depend on a number of surrounding water molecules, we came to the conclusion that a hydrogen-bonded cluster of n(H2O) greater than or equal to 41 water molecules could be considered as an appropriate model for simulation of ATI hydration effects.